2D Nuclear Magnetic Resonance Spectroscopy Research Articles

Anti-inflammatory properties of novel galloyl glucosides isolated from the Australian tropical plant Uromyrtus metrosideros

Two new galloyl glucosides, galloyl-lawsoniaside A (4) and uromyrtoside (6), were isolated from the polar fraction of Uromyrtus metrosideros leaf extract along with another four previously identified phytochemicals (1, 2, 3, and 5). The structures of these six compounds were characterised using low and high-resolution mass spectrometry (L/HRMS) and 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy. These compounds were not toxic to human peripheral blood mononuclear cells (PBMCs) at 10 μg/mL over 24 h, yet showed significant in vitro suppression of proinflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Specifically, the release of interferon γ (IFN-γ), interleukin (IL)-17A, and IL-8 from phorbol myristate acetate/ionomycin (P/I) and anti-CD3/anti-CD28-activated cells were significantly suppressed by compounds 4 and 5. Interestingly, no effect on tumour necrosis factor (TNF) release was observed. These results show that the newly characterised compound 4 has promising cytokine suppressive properties, which could be further investigated as a candidate for IBD treatment.

Synthesis and evaluation of benzoylbenzofurans and isoflavone derivatives as sirtuin 1 inhibitors with antiproliferative effects on cancer cells

Isoflavone derivatives were prepared from benzoylbenzofuran precursors. The synthesized compounds were analyzed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, as well as high-resolution mass spectrometry (HRMS) to confirm their structures. The benzoylbenzofuran and isoflavone analogues were evaluated for inhibition of sirtuin 1 (SIRT1) and cell proliferation in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Several isoflavone and benzoylbenzofuran derivatives exhibited potent antiproliferative effects against the MDA-MB-231 cancer cell line. Most of the isoflavone derivatives attenuated SIRT1 activity to below 50%. The most active compounds were the isoflavone quinones 38, 39, and 40, at IC50 values of 5.58 ± 0.373, 1.62 ± 0.0720, and 7.24 ± 0.823 μM, respectively. Importantly, the most active compound, 6-methoxy-4′,6′-dimethylisoflavone-2′,5′-quinone (39) displayed SIRT1 inhibitory activity comparable to that of the reference compound, suramin. The in silico docking simulations in the active site of SIRT1 further substantiated the experimental results and explored the binding orientations of potent compounds in the active site of the target.

Antimicrobial and Cytotoxic Cyathane-Xylosides from Cultures of the Basidiomycete Dentipellis fragilis.

In our continued search for biologically active metabolites from cultures of rare Basidiomycota species, we found eight previously undescribed cyathane-xylosides from submerged cultures of Dentipellis fragilis, which were named dentifragilins A–H. In addition, the known cyathane derivatives striatal D and laxitextine A were isolated. All compounds were characterized by high-resolution electrospray ionization mass spectrometry (HR-ESIMS) as well as by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Several of the compounds exhibited significant activities in standardized cell-based assays for the determination of antimicrobial and cytotoxic effects. The discovery of cyathanes in the genus Dentipellis has chemotaxonomic implications, as this class of diterpenoids has already been shown to be characteristic for mycelial cultures of the related genera Hericium and Laxitextum, which are classified as Dentipellis in the family Hericiaceae.

Simplex centroid mixture design, DI-ESI-MS, and chemometric analysis-guided isolation of parinarioidin C from bark of Brosimum parinarioides (Moraceae)

Brosimum parinarioides , a plant species native to the Amazonian region, is used in folk medicine for the treatment of several diseases. However, knowledge regarding its phytochemical content is scarce. In order to investigate the phytochemical composition of B. parinarioides , an integrative approach based on simplex-centroid design, direct infusion electrospray ionization mass spectrometry (DI-ESI-MS), and chemometric analysis was adopted. According to simplex-centroid design, dichloromethane (DCM), ethyl acetate (AcOEt), and ethanol-based (EtOH) extracts were prepared and analyzed using DI-ESI-MS, and their chemical profile was submitted to principal component analysis (PCA) and hierarchical cluster analysis (HCA). The AcOEt extract from the bark was obtained on a large scale and subjected to chromatographic separation. The multivariate analysis indicated the formation of three groups and highlighted isoliquiritigenin ( 1 ), licoagrochalcone A ( 2 ), caffeic acid glucoside ( 3 ), epicatechin-glycoside ( 4) , caffeic acid ( 5 ), and one unknown compound, as being responsible for the segregation of these groups. Thus, the new compound was isolated and characterized using 1D and 2D nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) as parinarioidin C ( 10 ). In addition, parinarioidin A ( 6 ), parinarioidin B ( 7 ), 3- O -caffeoylquinic acid ( 8 ), and kanzonol C ( 9 ) were tentatively identified based on manual interpretation of MS/MS spectra. Overall, the integrative approach proposed in the study provided a simple and effective model to dereplicate known compounds and guide the isolation of a new compound from a native amazon species, thus improving the knowledge regarding the phytochemical content of B. parinarioides . • One new compound was isolated from Brosimum parinarioides. • Nine phenolic compounds were identified based on the MS/MS data. • This study provided an effective model to guide the isolation of new natural products.

Conformational Properties and Putative Bioactive Targets for Novel Thiosemicarbazone Derivatives.

The structure assignment and conformational analysis of the thiosemicarbazones, DKI21 and DKI24, were performed through homonuclear and heteronuclear 2D Nuclear Magnetic Resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-ROESY, 2D-HSQC, and 2D-HMBC) and quantum mechanics (QM) calculations, using Functional Density Theory (DFT). In addition, utilizing a combination of 2D-NOESY and 2D-ROESY spectra an exo structure was established for both of the analogs. This experimental results were confirmed by theoretical mechanistic studies, as the lowest minima conformations derived through DFT calculations were compatible with the spatial correlations observed in the 2D-NOESY and 2D-ROESY spectra. Finally, molecular binding experiments were performed to detect the potential targets for DKI21 and DKI24, derived from SwissAdme. In silico molecular binding experiments showed favorable binding energy values for the most of the enzymes studied. The ADMET calculations, using the preADMET and pKCSm software, showed that the two molecules appear as possible drug leads.

Machine Learning Optimization of Lignin Properties in Green Biorefineries

Novel biorefineries could transform lignin, an abundant biopolymer, from side-stream waste to high-value-added byproducts at their site of production and with minimal experiments. Here, we report the optimization of the AquaSolv omni biorefinery for lignin using Bayesian optimization, a machine learning framework for sample-efficient and guided data collection. This tool allows us to relate the biorefinery conditions like hydrothermal pretreatment reaction severity and temperature with multiple experimental outputs, such as lignin structural features characterized using 2D nuclear magnetic resonance spectroscopy. By applying a Pareto front analysis to our models, we can find the processing conditions that simultaneously optimize the lignin yield and the amount of β-O-4 linkages for the depolymerization of lignin into platform chemicals. Our study demonstrates the potential of machine learning to accelerate the development of sustainable chemical processing techniques for targeted applications and products.

High-performance thin-layer chromatography – antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.)

The present work introduces a high-performance thin-layer chromatography (HPTLC)–direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC–EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC–mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform – ethyl acetate – methanol 15:3:2, V/V/V) was changed (to n-hexane – isopropyl acetate – methanol – acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1–2 and 3–4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50) values in the range of 32.3–64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

Structural characterization and antioxidant and anti-inflammatory activities of new chemical constituent from the seeds of bambara groundnut (Vigna subterranea (L.) Verdc.)

ABSTRACT A new compound (C20H18O9) was isolated from bambara groundnut seeds (Vigna subterranea (L.) Verdc.) and identified as a luteolin glycoside based on 1H, 13C and 2D nuclear magnetic resonance spectroscopy data and previously reported data. This compound exhibited similar scavenging activity for DPPH radical to Trolox and had a higher inhibitory effect on superoxide anion radicals than gallic acid and Trolox. C20H18O9 exhibited an inhibitory effect on DPPH and superoxide anion radicals, with IC50 values of 2.25 ± 0.14 µg/mL and 9.8 ± 0.3 µg/mL, respectively. However, C20H18O9 displayed low activities for FRAP and metal chelation. C20H18O9 had a higher inhibitory effect on lipoxygenase (IC50 = 2.16 ± 0.14 µg/mL) than nordihydroguaiaretic acid but had a lower inhibitory effect on xanthine oxidase than allopurinol. These results indicate that bambara groundnut seeds can be natural sources of antioxidants for humans and can serve as lipoxygenase inhibitor to reduce inflammation.

Eupholaricanone, a potent α-glucosidase anthracene derivative from Euphorbia larica Boiss

A potent α-glucosidase anthracene derivative (eupholaricanone, 1), along with three known compounds lupeol (2) and lupeol acetate (3), and lup 20(29)-ene (4) was isolated as a natural product from ethyl acetate fraction of Euphorbia larica. The structure of the compound was elucidated by 1D (1H- and 13C) and 2D (HSQC, HMBC, COSY and NOESY) nuclear magnetic resonance (NMR) spectroscopy and mass (QTOF-LCMS) spectrometry, and crystal structure was determined by single-crystal X-ray diffraction analysis. In vitro, compound 1 displayed potent α-glucosidase inhibitory activity with 91.60% inhibition and IC50 value of 45.91 ± 0.61 µM. Furthermore, in silico structural bioinformatics tool was employed to predict the binding pattern of compound 1 in the active site of α-glucosidase where compound demonstrated excellent binding interactions with Arg213 and His351 and exhibited higher docking score than the substrate molecule. Our experimental findings and docking results indicate that this anthracene derivative could serve as a lead compound upon optimization.

Conformational Properties of New Thiosemicarbazone and Thiocarbohydrazone Derivatives and Their Possible Targets.

The structure assignment and conformational analysis of thiosemicarbazone KKI15 and thiocarbohydrazone KKI18 were performed through homonuclear and heteronuclear 2D Nuclear Magnetic Resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-HSQC, and 2D-HMBC) and quantum mechanics (QM) calculations using Functional Density Theory (DFT). After the structure identification of the compounds, various conformations of the two compounds were calculated using DFT. The two molecules showed the most energy-favorable values when their two double bonds adopted the E configuration. These configurations were compatible with the spatial correlations observed in the 2D-NOESY spectrum. In addition, due to the various isomers that occurred, the energy of the transition states from one isomer to another was calculated. Finally, molecular binding experiments were performed to detect potential targets for KKI15 and KKI18 derived from SwissAdme. In silico molecular binding experiments showed favorable binding energy values for all four enzymes studied. The strongest binding energy was observed in the enzyme butyrylcholinesterase. ADMET calculations using the preADMET and pKCSm software showed that the two molecules appear as possible drug leads.

Phloroglucinol–meroterpenoids from the leaves of Eucalyptus camaldulensis Dehnh.

Fourteen undescribed phloroglucinol-meroterpenoids, namely eucalypcamals A–N, were isolated from a CH2Cl2 extract of the leaves of Eucalyptus camaldulensis Dehnh. In addition, from the same extract, twelve known phloroglucinols, three known flavonoids, and four known phenolic compounds were also isolated. The structures of the undescribed compounds were analyzed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, and high resolution electrospray ionization mass spectrometry (HRESIMS). The assignments of the absolute configurations were performed by comparing the experimental electronic circular dichroism (ECD) data with the calculated values. Eucalyprobusal E was found to be cytotoxic against HCT116, Jurkat, and MDA-MB-231 cell lines with IC50 values of 17.6, 9.44, and 17.9 μM, respectively. Eucalrobusone F exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA) and S. aureus with minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) values of 4/4 μg/mL while euglobal Ia1 showed antifungal activity with MIC/MFC values of 16/16 μg/mL.

Cembranoids From the Leaves of Nicotiana tabacum and Their Neuroprotective Activities

A new cembrane-type diterpenoid (1) and 9 known cembranoids (2-10) were isolated from the leaves of Nicotiana tabacum. The structure of the new metabolite was confirmed by extensive analyses of their spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D- and 2D nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy (IR). All of the isolated compounds were tested for their protective effects against oxygen-glucose deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells. The new compound 1 exhibited a moderate protective effect against OGD-induced neurotoxicity at 10 μM.

Secondary Metabolites from Hericium erinaceus and Their Anti-Inflammatory Activities.

Hericium erinaceus, a culinary and medicinal mushroom, is widely consumed in Asian countries. Chemical investigation on the fruiting bodies of Hericium erinaceus led to the isolation of one new ergostane-type sterol fatty acid ester, erinarol K (1); and eleven known compounds: 5α,8α -epidioxyergosta-6,22-dien-3β-yl linoleate (2); ethyl linoleate (3); linoleic acid (4); hericene A (5); hericene D (6); hericene E (7); ergosta-4,6,8(14),22-tetraen-3-one (8); hericenone F (9); ergosterol (10); ergosterol peroxide (11); 3β,5α,6α,22E-ergosta-7,22-diene-3,5,6-triol 6-oleate (12). The chemical structures of the compounds were determined by 1D and 2D NMR (nuclear magnetic resonance) spectroscopy, mass spectra, etc. Anti-inflammatory effects of the isolated aromatic compounds (5–7, 9) were evaluated in terms of inhibition of pro-inflammatory mediator (TNF-α, IL-6 and NO) production in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage cells. The results showed that compounds 5 and 9 exhibited moderate activity against TNF-α (IC50: 78.50 μM and 62.46 μM), IL-6 (IC50: 56.33 μM and 48.50 μM) and NO (IC50: 87.31 μM and 76.16 μM) secretion. These results supply new information about the secondary metabolites of Hericium erinaceus and their anti-inflammatory effects.

Steroidal saponins with anti-inflammatory activity from Tribulus terrestris L.

Abstract Objective: Tribulus terrestris L. (T. terrestris) is a highly valuable traditional Chinese medicine used to treat stroke, inflammation, pulmonary fibrosis, liver cancer, and urolithiasis. To identify the basic substance responsible for the anti-inflammatory effect of TST (total saponins of Tribulus), its chemical composition was systematically studied, and its effect of inhibiting nitric oxide generation and the expression of related inflammatory factors were determined. Methods: To separate chemical constituents from T. terrestris by column chromatography. Spectroscopic methods, including 1D and 2D nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) techniques, were used to elucidate the isolated compounds. The anti-inflammatory activities of TST and several compounds were evaluated in vitro. Results: Fifteen steroidal saponins, including 9 furostanol steroidal saponins (1, 2, 3, 4, 5, 6, 7, 8, and 15) and 6 isospirostanol steroidal saponins (9, 10, 11, 12, 13, and 14), were isolated from T. terrestris. TST significantly decreased the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in RAW 264.7 cells stimulated by lipopolysaccharides. Compounds 13 and 15 evidently reduced TNF-α expression. Compounds 6, 10, 12, 13, and 15 markedly reduced IL-6 expression. Conclusions: Compounds 1 was a novel furostanol steroidal saponin, named 26-O-β-D-glucopyranosyl-(25R)-5α-furostan-12-carbonyl-20(22)-en-3β, 26-diol-3-O-{β-D-xylopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside}. Compounds 2 was isolated from the family Zygophyllaceae for the first time, and 5 and 6 were isolated from the Tribulus genus. TST and compounds 6, 10, 12, 13, and 15 exerts anti-inflammatory activity.

Terminalin from African Mango (Irvingia gabonensis) Stimulates Glucose Uptake through Inhibition of Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs), along with protein tyrosine kinases, control signaling pathways involved in cell growth, metabolism, differentiation, proliferation, and survival. Several PTPs, such as PTPN1, PTPN2, PTPN9, PTPN11, PTPRS, and DUSP9, disrupt insulin signaling and trigger type 2 diabetes, indicating that PTPs are promising drug targets for the treatment or prevention of type 2 diabetes. As part of an ongoing study on the discovery of pharmacologically active bioactive natural products, we conducted a phytochemical investigation of African mango (Irvingia gabonensis) using liquid chromatography–mass spectrometry (LC/MS)-based analysis, which led to the isolation of terminalin as a major component from the extract of the seeds of I. gabonensis. The structure of terminalin was characterized by spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution (HR) electrospray ionization (ESI) mass spectroscopy. Moreover, terminalin was evaluated for its antidiabetic property; terminalin inhibited the catalytic activity of PTPN1, PTPN9, PTPN11, and PTPRS in vitro and led to a significant increase in glucose uptake in differentiated C2C12 muscle cells, indicating that terminalin exhibits antidiabetic effect through the PTP inhibitory mechanism. These findings suggest that terminalin derived from African mango could be used as a functional food ingredient or pharmaceutical supplement for the prevention of type 2 diabetes.

Inversion of Supramolecular Chirality by In Situ Hydrolyzation of Achiral Diethylene Glycol Motifs.

Chiral inversion of supramolecular assemblies is of great research interest due to its broad practical applications. However, chiral structure transition induced by in situ regulation of building molecules has remained a challenge. Herein, left-handed fibrous assemblies were constructed by C2-symmetic l-phenylalanine coupled with diethylene glycol (LPFEG) molecules. In situ hydrolyzing terminal diethylene glycol motifs in LPFEG successfully inverted the chirality of the nanofibers from left- to right-handedness. The transition of right-handed fibers into left-handed fibers could also be achieved via hydrolyzing DPFEG molecules. Circular dichroism (CD) spectroscopy, 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy revealed that the back-folded achiral diethylene glycol played a vital role in L/DPFEG molecular arrangements and removing terminal diethylene glycol could induce the opposite rotation of molecular assemblies. Thanks to this merit, the enantioselective separation of racemic phenylalanine was obtained and the enantiomeric excess (ee) values could achieve around ±20% after separation. This study not only provides a new strategy to regulate the chiral structure via dynamic modulation of terminal substituents but also presents a promising application in the field of enantioselective separation.

Two New Fatty Acid Derivatives, Omphalotols A and B and Anti-Helicobacter pylori Fatty Acid Derivatives from Poisonous Mushroom Omphalotus japonicus.

As part of ongoing systematic research into the discovery of bioactive secondary metabolites with novel structures from Korean wild mushrooms, we investigated secondary metabolites from a poisonous mushroom, Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. belonging to the family Marasmiaceae, which causes nausea and vomiting after consumption. The methanolic extract of O. japonicus fruiting bodies was subjected to the fractionation by solvent partition, and the CH2Cl2 fraction was analyzed for the isolation of bioactive compounds, aided by an untargeted liquid chromatography mass spectrometry (LC–MS)-based analysis. Through chemical analysis, five fatty acid derivatives (1–5), including two new fatty acid derivatives, omphalotols A and B (1 and 2), were isolated from the CH2Cl2 fraction, and the chemical structures of the new compounds were determined using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high resolution electrospray ionization mass spectrometry (HR-ESIMS), as well as fragmentation patterns in MS/MS data and chemical reactions followed by the application of Snatzke’s method and competing enantioselective acylation (CEA). In the anti-Helicobacter pylori activity test, compound 1 showed moderate antibacterial activity against H. pylori strain 51 with 27.4% inhibition, comparable to that of quercetin as a positive control. Specifically, compound 3 exhibited the most significant antibacterial activity against H. pylori strain 51, with MIC50 and MIC90 values of 9 and 20 μM, respectively, which is stronger inhibitory activity than that of another positive control, metronidazole (MIC50 = 17 μM and MIC90 = 46 μM). These findings suggested the experimental evidence that the compound 3, an α,β-unsaturated ketone derivative, could be used as a moiety in the development of novel antibiotics against H. pylori.

Hemoglobin–Laccase Modifications of Ligninsulfonate: A Promising Synergistic Stratagem for Lignin Biodegradation

AbstractMulticomponent systems, such as laccase cooperating with other proteins in ligninase, are able to modify the chemical structure of lignin efficiently and opens the door to potential applications. Here, an in‐depth investigation of hemoglobin‐assisted laccase oxidation of soluble lignosulfonate is carried out, using a combination of UV–visible spectroscopy, isothermal titration calorimetry, gel permeation chromatography, Raman spectroscopy, 2D nuclear magnetic resonance spectroscopy, and high‐performance liquid chromatography–mass spectrometry. Intriguingly, hemoglobin enhances the exothermically oxidative process of the laccase reaction. Product characterizations suggest that hemoglobin may preferentially reduce the ratio of guaiacyl/syringyl subunits and promote phenylcoumaran formation simultaneously. The complicated interactions among hemoglobin, laccase, and lignin make a multiple‐component reaction, suggesting that Fe‐containing macromolecules have the capacity to mediate laccase based chemical modifications of lignin.

Poloxamer-enhanced solubility of griseofulvin and its related antifungal activity against Trichophyton spp.

Poorly water-soluble drugs, such as the antifungal drug griseofulvin (GF), exhibit limited bioavailability, despite their high membrane permeability. Several technological approaches have been proposed to enhance the water solubility and bioavailability of GF, including micellar solubilization. Poloxamers are amphiphilic block copolymers that increase drug solubility by forming micelles and supra-micellar structures via molecular self-association. In this regard, the aim of this study was to evaluate the water solubility increment of GF by poloxamer 407 (P407) and its effect on the antifungal activity against three Trichophyton mentagrophytes and two T. rubrum isolates. The GF water solubility profile with P407 revealed a non-linear behavior, well-fitted by the sigmoid model of Morgan-Mercer-Flodin. The polymer promoted an 8-fold increase in GF water solubility. Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and 2D nuclear magnetic resonance (NMR Roesy) spectroscopy suggested a GF-P407 interaction, which occurs in the GF cyclohexene ring. These results were supported by an increase in the water solubility of the GF impurities with the same molecular structure. The MIC values recorded for GF ranged from 0.0028 to 0.0172 mM, except for T. Mentagrophytes TME34. Notably, the micellar solubilization of GF did not increase its antifungal activity, which could be related to the high binding constant between GF and P407.

Isolation and quantification of diarylheptanoids from European hornbeam (Carpinus betulus L.) and HPLC-ESI-MS/MS characterization of its antioxidative phenolics

Detailed polyphenol profiling of European hornbeam (Carpinus betulus L.) bark, leaf, male and female catkin extracts was performed by high-performance liquid chromatography–diode array detection coupled to tandem mass spectrometry (HPLC-DAD-MS/MS). A total of 194 compounds were characterized and tentatively identified. Gallo- and ellagitannins dominated in the methanol extracts, while flavonol glycosides and methoxylated flavones prevailed in the ethyl acetate samples. In the quest for diarylheptanoids, twelve compounds were isolated by the combination of subsequent reversed-phase flash chromatographic and high-performance liquid chromatographic methods. The structural elucidation of the isolated components was performed by ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (UHPLC‐Orbitrap‐MS) as well as 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Six known cyclic diarylheptanoids, together with a new compound were described in Carpinus betulus for the first time. The occurrence of a linear diarylheptanoid and a lignan has also been unprecedented in the genus Carpinus. Moreover, three known flavonol glycosides were isolated. Based on the identification of characteristic fragment ions, a new mass spectrometric fragmentation pathway for meta,meta-cyclophane-type diarylheptanoids was proposed. Quantities of the four major cyclic diarylheptanoids in European hornbeam were determined by a validated UHPLC-DAD method for the first time. The antioxidant properties of the extracts and the isolated compounds were assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Contribution of the individual constituents to the total radical scavenging activity of the samples was evaluated by an off-line DPPH-HPLC-DAD method. This allowed the identification of gallo- and ellagitannin derivatives as the constituents being primarily responsible for the antioxidant capacity of the extracts.